Insulin-Like Growth Factor Binding Protein-Related Protein 1 Mediates VEGF-Induced Proliferation, Migration and Tube Formation of Retinal Endothelial Cells

Abstract

The potential role of insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) as an endogenous angiogenesis inhibitor in the prevention of vascular endothelial growth factor (VEGF)-induced retinal angiogenesis has not been explored. Expression of IGFBP-rP1 in rhesus macaque choroid-retinal endothelial cell line (RF/6A) cells was assessed by reverse transcription polymerase chain reaction analysis and Western blotting. RF/6A cells were treated with VEGF (10 ng/ml) alone or in the presence of IGFBP-rP1 at concentrations ranging from 50 ng/ml to 200 ng/ml. The proliferation, migration and capillary-like tube formation of RF/6A cells were evaluated using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium colorimetric assay, the chemotactic motility assay and the Matrigel tube formation assay, respectively. Flow cytometry was used to detect the apoptosis of RF/6A cells, and oncogenic B-Raf expression was assessed to elucidate the pathway for IGFBP-rP1-mediated induction of apoptosis in the presence of VEGF. RF/6A cells expressed both IGFBP-rP1 transcripts and IGFBP-rP1 protein. VEGF markedly stimulated proliferation, migration and capillary-like tube formation of RF/6A cells (P<0.05), whereas those VEGF-induced parameters were significantly inhibited by IGFBP-rP1 at concentrations ranging from 50 ng/ml to 200 ng/ml in a dose-dependent manner (P < 0.05). Following addition of IGFBP-rP1, expression of B-Raf was significantly decreased dose-dependently, and apoptosis occurred as evidenced by flow cytometry (P < 0.05). IGFBP-rP1 can inhibit the stimulatory effect of VEGF on retinal angiogenesis in vitro by inhibiting expression of B-Raf to induce apoptosis. It is a novel endogenous anti-angiogenic factor with potential therapeutic action in retinal neovascularization dependent disorders.